Aging is a complex process driven by diverse molecular pathways and biochemical events. A detailed understanding of the molecular and cellular events influencing longevity remains a central question in the biology of aging. The emergence of model systems to study aging and the use of whole genome approaches are helping us decipher these processes. In efforts to identify novel genes involved in longevity, several methods using C. elegans as a model have been employed, including genome wide RNAi screens, DNA microarrays, and mass spectrometry methods. While a handful of E3 ubiquitin ligases have been implicated in longevity pathways based on these screens, the mechanisms by which these ligases function have not been identified. Recently, several E3 ligases have been implicated in regulating longevity in C. elegans, including rnf-5, K08D12.3, lin-23, and phi-3. We also have results showing that the ubiquitin ligase wwp-1 is a positive regulator of lifespan in C. elegans. We have optimized and validated powerful assays that will provide excellent opportunities to study the functions of ubiquitin ligases controlling aging. These assays involve TAP purification and a new enzymatic/immunopurification strategy that enriches for ubiquitinated substrates, facilitating the identification of E3 ligase substrates by mass spectrometry. These approaches have been employed successfully by our team to identify novel substrates for F-box-containing E3 ligases. We plan on using similar strategies to identify substrates of worm and human ubiquitin ligases that our preliminary results, together with published findings, suggest to be involved in aging. We will confirm that proteins identified in our purification screens are bona fide targets that regulate longevity using binding assays, siRNA approaches, in vitro ubiquitination assays, and lifespan analysis in C. elegans. Many chronic and degenerative diseases, such as cancer, cardiovascular disease, diabetes, and neurodegenerative diseases, develop in an age-related manner. A detailed understanding of the pathways that regulate longevity may lead to novel therapies for age-related diseases. Identifying substrates of ubiquitin ligases will provide important insights into human aging and the development of age-related pathology and disease. Under this collaborative project, we will systematically identify substrates of human ubiquitin ligases involved in the regulation of longevity (Specific Aim 1). Under Aim 2, we will contribute to validate the biologically most significant substrates identified under Aim 1. The research and results obtained under this award will be important for human health as it will identify new drug targets to combat most age related diseases, including Cancer and Alzheimer's Disease.